Salts of a glutathione alkylester and anaminoacids

ABSTRACT

Crystalline non-hygroscopic salt of glutathione alkylester of the formula (I) ##STR1## wherein AA is an amino acid are disclosed.

This application is filed under U.S.C. 371 of the application PCT/EP93101462 filed 6/9/93.

The present invention concerns new salts of the L-γ-glutamyl-L-cysteinylalkyl esters.

More particularly, the invention relates to the salts with amino acidsof glutathione (C₁ -C₆) alkylesters and to their solvates. The inventionfurther relates to a process for the preparation of these new salts andto pharmaceutical compositions containing such salts as activeingredient.

Glutathione, L-γ-glutamyl-L-cysteinylglycine or GSH, is represented bythe formula (A) ##STR2## Glutathione, a tripeptide containing an SHgroup, is well known as a cellular reducer, as a catalyst in a greatnumber of biological reactions, as a metabolic reagent, and as a form ofcysteine storage and control.

Glutathione plays a very important role in cell protection against freeradicals, against reactive oxygenated species such as hydroperoxides andperoxides, and against toxic compounds of either endogenous andhexogenous origin (A. Meister et al., Ann. Rev. Biochen 52,711,1983).

An increase of intracellular GSH may therefore induce cell protectionagainst damage due to radiation, or to toxic chemical species, such asendogenous peroxides or highly toxic drugs.

Glutathione as such, however, cannot pass the cellular barrier andconsequently does not enter cells, while its esters do. Moreparticularly its ethylester (GSEt) is capable of penetrating the cells,thus undergoing in situ hydrolization (M. E. Anderson et al. Arch.Biochem. Biophys. 1985,239,538-538; U.S. Pat. No. 170,489).

Glutathione ethylester (on glycine) is prepared according to the methoddescribed in the publication by M. E. Anderson et al. mentioned above,but the final product always contains a certain amount of diester (onboth glutamic acid and glycine) which must be eliminated as it is toxic.

This impurity can be removed by chromatography on weak cation exchangeresin columns, as described in U.S. Pat. 4,710,489, but such anoperation is not easy, when it has to be performed on an industrialscale.

U.S. Pat. No. 4,709,013 describes glutathione ester sulphates highlycrystallizable and therefore easily purifiable, allowing their easytransformation into glutathione monoester devoid of the toxic diester.

Although the sulphates described in the above document arecrystallizable, the thus obtained crystals are hygroscopic. Theusefulness of such sulphates is therefore limited to their use asintermediates in the purification of glutathione esters.

Consequently, these sulphates cannot be used as active ingredients ofpharmaceutical compositions, due to the difficulties encountered intheir handling in pharmaceutical technique.

Another reason which hinders said sulphates from being used asmedicaments is their high acidity.

Patent EP 257 992 describes the use of glutathione monoesters for thepreparation of agents suitable for use in the prophylaxis and treatmentof cerebral ischemia and, as active ingredients, it reports alsoinorganic salts such as hydrochlorides, nitrates, or organic ones suchas oxalates, p-toluensulphonates, maleates, etc. In the above documentit is however pointed out that when such monoesters are beingadministered in the form of salts, they are previously desalted, oradministered by concurrent addition of a base, i.e. sodium carbonate,neutralizing them. Consequently, glutathione esters stable crystallinesalts directly useful as active ingredients for pharmaceuticalcompositions are not known in literature.

It has now surprisingly been found that glutathione monoalkylesters,namely the esters of a tripeptide with an initial alpha-aminoacid unit,easily yield salts with other aminoacids.

Moreover, it has been found that these salts with aminoacids arecrystalline and not hygroscopic.

Finally, it has been found that the above reported crystalline nonhygroscopic salts of glutathione alkylesters are even pharmacologicallymore active that the free base, and are easy to handle in pharmaceuticaltechnique for the preparation of compositions for therapeutic use.

Thus, it is an object of the present invention to provide crystallinenon hygroscopic salts of glutathione alkylesters (GSAlk) with aminoacidsof formula (I) ##STR3## wherein AA is the salifying aminoacid and Alkrepresents an alkyl group containing from 1 to 6 carbon atoms such asmethyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, n-pentyl, isoamyl,n-hexyl.

Advantageous salts of glutathione alkylesters are those of glutathioneethylester (GSTt) represented by the formula (I') ##STR4## wherein AA isas defined above, and those of glutathione isopropyl ester (GSIpr)represented by the formula (I") ##STR5## wherein AA is as defined above.

There are mentioned, among the salifying aminoacids giving crystallinenon hygroscopic salts, natural alpha-aminoacids such as glycine,alanine, aspartic acid and glutamic acid, but other salifying aminoacidssuch as pyroglutamic acid, γ-aminobutyric acid and taurine also provideconsiderable advantages.

As compared with sulphate and hydrochloride, namely with the saltsspecifically described in literature, the new GSAlk salts of the presentinvention, besides the advantage of being crystalline, stable and nothygroscopic, they have also the advantage of being physiologicallycompatible, being less acid. In fact, in aqueous solution, they providea physiologically acceptable pll.

In this connection, particularly preferred GSAlk according to thepresent invention are those having the formula (I) above, in which AA isselected from the group consisting of taurine, γ-aminobutyric acid,pyroglutamic acid, glycine, alanine, aspartic acid and glutamic acid andAlk is ethyl (formula I' above) or isopropyl (formula I" above).

Moreover, while the GSEt sulphate described in U.S. Pat. No. 4,709,013crystallizes with some difficulty and in a modest yield, the salts ofthe present invention are easily prepared by precipitation and areobtained in extremely high yields.

Thus, according to another of its aspects, the present inventionconcerns a process for the preparation of crystalline non hygroscopicsalts of glutathione alkylester (I). which comprises:

a) treating said glutathione alkylester with the adequate aminoacid inwater

b) evaporating the solvent under reduced pressure till a small volume

c) treating the reaction mixture with an alcohol and isolating theprecipitated salt.

Salification is carried out at a temperature ranging from 0° to 40° C.,during a period of time varying from 1 to 6 hours, and ethyl alcohol isused by preference as the precipitating alcohol. The thus obtained saltis isolated by simple filtration of the precipitate. If necessary, saidprecipitate may be washed with cold ethyl alcohol or with anotherorganic solvent.

The aminoacid which forms the GSAlk salt is preferably used inequimolecular amount in respect of the starting GSAlk.

The thus obtained new salts may contain some crystallization water ofethyl alcohol. Said solvates are within the scope of the presentinvention.

GSAlk salts and their solvates increase the stability and the solubilityof GSAlk, thus increasing glutathione intracellular levels.

Consequently they are useful as therapeutically active agents indetoxication from drugs, in cellular protection versus peroxides, freeradicals and radiations.

Besides, the compounds of the present invention may be used to preventcellular damage due to ischemia and/or hypoxia.

Thus, it is another object of the present invention to providepharmaceutical compositions containing as active ingredient, at leastone of the crystalline non-hygroscopic salts of GSAlk with aminoacids orof their solvates in admixture with a pharmaceutical carrier.

The pharmaceutical compositions of the present invention contain from 10to 1000 mg of active ingredient calculated on the basis of glutathionealkylester, by preference from 20 to 500 mg of equivalent in glutathionealkylester per dose unit They can be prepared in form of vials or tinybottles for intravenous or intramuscular injection, as suppositories orrectal capsules, in forms for the oral administration, such as tablets,capsules or granulates, or as pharmaceutical form suitable for nasaladiministration in nebulizers.

The carriers are those of common use in pharmaceutical technique forpreparing oral, parenteral, rectal, transdermal or intranasalformulations, provided they are chemically compatible with the active orinert ingredients.

The following non-limiting examples illustrate the invention.

EXAMPLE 1 GSEt taurine salt (compound 1)

Equimolar quantities of GSEt (4.8 g) and of taurine (1.81 g) weredissolved in H₂ O (80 ml) at room temperature. The solvent wasevaporated to small volume in vacuum. The product was allowed tocrystallize after adding 100 ml of ethyl alcohol. Filtration and washingwith a small quantity of cold ethyl alcohol were done Drying in oven at40° C. in vacuum was then performed, obtaining 5.91 g.

m.p.=140°-145° C.

Calculated: C=36.51; H=6.13; N=12.17. Found: C=36.55; H=6.12; N=12.20.

HPLC assay=73% (GSEt); 27% (Taurine)

EXAMPLE 2 GSEt γ-aminobutyric acid salt (compound 2)

Equimolar quantities of GSEt (4.8 g) and of γ-aminobutyric acid (1.49 g)were dissolved in H₂ O (60 ml) at room temperature. The solvent wasevaporated to small volume, ethyl alcohol (100 ml) was added and theproduct was allowed to crystallize. Filtration and washing with coldethyl alcohol were done. Drying in oven at 40° C. in vacuum was thenperformed, obtaining 5.85 g.

m.p.=124°-127° C.

Calculated: C=43.82; H=6.90; N=12.78. Found: C=43.79; H=6.89; N=12.80.

HPLC assay=76.48% (GSEt); 23.52% (γ-aminobutyric acid)

EXAMPLE 3 GSEt pyroglutamic acid salt (compound 3)

Equimolar quantities of GSEt (4.8 g) and of pyroglutamic acid (1.86 g)were dissolved in H₂ O (60 ml) at room temperature. The solvent wasevaporated to small volume, ethyl alcohol (100 ml) was added and theproduct was allowed to crystallize. Filtration and washing with coldethyl alcohol were done. Drying in oven at 40° C. in vacuum was thenperformed, obtaining 3.50 g.

m.p.=130°-133° C.

Calculated: C=43.96; H=6.07; N=12.06. Found: C=43.94; H=6.08; N=12.08.

HPLC assay=72.2% (GSEt); 27.8% (pyroglutamic acid)

EXAMPLE 4 GSEt glycine salt (compound 4)

Equimolar quantities of GSEt (4.8 g) and of glycine (1.07 g) weredissolved in H₂ O (70 ml) at room temperature. The solvent wasevaporated to small volume, ethyl alcohol (100 ml) was added and theproduct was allowed to crystallize.

Filtration and washing with cold ethyl alcohol were done. Drying in ovenat 40° C. in vacuum was then performed, obtaining 4.80 g.

m.p.=161°-164° C.

Calculated: C=40.97; H=6.38; N=13.65. Found: C=40.93; H=6.37; N=13.67.

HPLC assay=81.7% (GSEt); 18.3% (glycine)

EXAMPLE 5 GSEt alanine salt (compound 5)

Equimolar quantities of GSEt (4.8 g) and of alanine (1.27 g) weredissolved in H₂ O (70 ml) at room temperature. The solvent wasevaporated to small volume, ethyl alcohol (100 ml) was added and theproduct was allowed to crystallize.

Filtration and washing with cold ethyl alcohol were done. Drying in ovenat 40° C. in vacuum was then performed, obtaining 4.65 g.

m.p.=163°-167° C.

Calculated: C=42.44; H=6.65; N=13.20. Found: C=42.40; H=6.66; N=13.18.

HPLC assay=79.01% (GSEt); 20.99% (alanine)

EXAMPLE 6

GSEt aspartic acid salt (compound 6)

Equimolar quantities of GSEt (4.8 g) and of aspartic acid (1.90 g) weredissolved in H₂ O (200 ml) at room temperature. The solvent wasevaporated to small volume, ethyl alcohol (100 ml) was added and theproduct was allowed to crystallize. Filtration and washing with coldethyl alcohol were done. Drying in oven at 40° C. in vacuum was thenperformed, obtaining 5 g.

m.p.=135°-138° C.

Calculated: C=41.02; H=6.02; N=11.96. Found: C=41.00; H=6.03; N=11.95.

HPLC assay=71.59% (GSEt); 28.41% (aspartic acid)

EXAMPLE 7 GSEt glutamic acid salt (compound 7)

Equimolar quantities of GSEt (4.8 g) and of glutamic acid (2.10 g) weredissolved in H₂ O (100 ml) at 50° C. The solvent was evaporated to smallvoulume, ethyl alcohol (100 ml) was added and the product was allowed tocrystallize.

Filtration and washing with cold ethyl alcohol were done. Drying in ovenat 40° C. in vacuum was then performed, obtaining 5.1 g.

m.p.=187°-190° C.

Calculated: C=42.31; H=6.26; N=11.61. Found: C=42.29; H=6.25; N=11.63.

HPLC assay=69.51% (GSEt); 30.49% (glutamic acid)

EXAMPLE 8 GSIpr taurine salt (compound 8)

12.4 g of glutathione were suspended in 80 ml of isopropanol, 4.2 ml ofH₂ SO₄ 95% were added dropwise. The solution became clear. Stirring wascontinued overnight. Crystals began to separate and after 24 hourscrystallization was finished. The solid was filtered off and washed with20 ml of ice-cooled isopropanol. Crystals were dissolved in 150 ml ofwater and then purified charging it into a column packed with 25 ml ofAmberlite IKC 68. The eluted fractions was added with an equimolarquantity of taurine then the solution was evaporated to drynessaffording 15.3 g (80%) of title compound.

HPLC assay=72% (GSIpr); theoretical=73.63%; assay=97.9%.

EXAMPLE 9 GSIpr taurine salt (compound 8)

12.4 g of glutathione were suspended in 100 ml ofisopropanol/hydrochloric acid 20%. The suspension became clear during 15minutes. Stirring was continued overnight. The solvent was evaporated todryness, 100 ml of water added and the pH was corrected to 8.5 withtriethylamine. The mixture was extracted 6 times with 50 ml of CH₂ Cl₂each time. The organic layer was discarded. An equimolar amount oftaurine was added to the water layer then the solution was evaporated todryness to afford 15.5 g (81%) of the title compound.

HPLC assay=70% (GSIpr); theoretical=73.63%; assay=95%.

EXAMPLE 10 Ampoules for intravenous or intramuscular injection

Composition:

    ______________________________________                                        Compound 2         30         mg                                              Sodium chloride FU IX                                                                            30         mg                                              Water for injection                                                                              5          ml                                              ______________________________________                                    

Compound 2 was dissolved in 5 ml of water for injection, sodium chloridewas added, filtration on 0.22μ membrane took place and the product wasintroduced in amber glass 5 ml ampoules. All operations were performedin a sterile environment.

EXAMPLE 11 Suppositories

Composition:

    ______________________________________                                        Compound 6         200        mg                                              Witepsol H 15 Ph.Eur.to                                                                          3          g                                               ______________________________________                                    

A suspension of Compound 6 in melted Witepsol and, by the suitablemachine, the 3 g suppository dies were filled. The whole was cooled.

EXAMPLE 12 Enteric-coated capsules

Composition:

    ______________________________________                                        Compound 1         200        mg                                              Starch             100        mg                                              Magnesium stearate 2          mg                                              ______________________________________                                    

The ingredients were mixed in a mixer and, by the suitable machine, theywere shared into enteric-coated type 1 capsule.

EXAMPLE 13 Nasal spray

Composition:

    ______________________________________                                        Compound 1         500        mg                                              Sodium chloride    50         mg                                              Purified water     10         ml                                              ______________________________________                                    

Sodium chloride and compound 1 were dissolved in water, and the productwas packaged in bottled equipped with 100 μl metering pump.

EXAMPLE 14 Enteric-coated tablets

Composition of core

    ______________________________________                                        Compound 3         100        mg                                              Lactose            60         mg                                              Starch             40         mg                                              Aerosyl            20         mg                                              Magnesium stearate 2          mg                                              ______________________________________                                    

Composition of coating

    ______________________________________                                               Eudragit L                                                                            3            mg                                                       Eudragit S                                                                            3            mg                                                ______________________________________                                    

The mixture was compressed directly thus obtaining the cores, that weresubsequently coated in a coating pan, by spraying the suspension ofEudragit L and Eudragit S in water.

We claim:
 1. A crystalline non-hygroscopic salt of a glutathionealkylester of the formula (I) ##STR6## wherein AA is taurine and Alk isethyl.
 2. A pharmaceutical composition containing, as active ingredient,a compound of claim 1 in admixture with a pharmaceutical excipient.
 3. Apharmaceutical composition according to claim 2, wherein the unit dosageof the active ingredient, calculated on the basis of the glutathioneester, is from 10 to 1000 mg.
 4. A pharmaceutical composition accordingto claim 3, wherein the unit dosage of the active ingredient is from 20to 500 mg.